人乳头瘤病毒16型E7基因原核表达载体的构建及表达

Construction and expression of human papillomavirus type-16 E7 gene in prokaryotic expressing vector

目的:构建人乳头瘤病毒16型(HPV-16)E7基因原核表达载体,观察目的基因的蛋白表达。方法:人工合成HPV16E7基因核苷酸序列,利用特异性引物通过聚合酶链反应扩增HPV16E7基因,并克隆至pGEX-4T1原核表达载体,构建重组表达载体pGEX-4T1-HPV16-E7。将其转化到工程菌Rosetta后,经IPTG诱导表达,进行E7-GST融合蛋白的SDS-PAGE分析和Ni 2+-NTA亲和层析纯化。结果:E7基因PCR扩增产物和重组表达质粒的双酶切产物经琼脂糖凝胶电泳,均可见300bp的目的基因条带。表达的重组蛋白经SDS-PAGE电泳和纯化后,在相对分子质量约36 000处有特异性表达带,与预期相符。结论:在原核系统中含E7基因的重组质粒可有效表达E7-GST融合蛋白。

Objective To construct prokaryotic expression vector harboring human papillomavirus-16 E7（HPV-16） gene and observe the E7 protein expression.Methods Synthetic E7 gene was amplified using polymerase chain reaction and subcloned into prokaryotic expressing vector pGEX-4T1.The recombinant plasmids,pGEX-4T1-HPV16-E7,were transferred into engineering bacteria Rosetta,and the fusion proteins E7-GST were analyzed on SDS-PAGE and purified by Ni2^＋-NTA affinity chromatography under induction with IPTG.Results The target gene fragment at a length of 300 bp was observed on the agarose gel electrophoresis pattern and PCR.SDS-PAGE analysis showed that the expressed recombinant protein,with a relative molecular mass of about 36 000,could be found.Conclusion The recombinant protein,E7-GST,is effectively expressed in prokaryotic expression system.